Mutants shikimic acid pathway

Rifamycins.—Contained within the rifamycin skeleton, for example that of rifamycin S (169), is a C7-N unit (heavy bonding) which has been deduced to arise from an intermediate of the shikimic acid pathway. Fresh evidence arising from a study using mutants of Nocardia meditenanei confirms this view, and it is apparent that divergence from the shikimate pathway to rifamycin synthesis occurs between sedoheptulose-7-phosphate and shikimic acid itself. 

Most steps of the shikimic acid pathway have been studied by means of mutants, isotopically labeled precursors, and studies of the specific enzymes from cell-free systems. The use of mutants for examination of various steps of the shikimic acid pathway has been of great value for the study of the enzymes and intermediates involved. Use of mutants permits interruption of the pathway at a stage that normally would not be seen. Mutants that involve one step of die pathway are especially useful. In some situations, intermediates are accumulated in large quantities, facilitating analysis of the sequence of steps in the pathways. Sometimes, however, the accumulation of intermediates exerts control over earlier stages of the biosynthetic process and inhibits the activity or represses formation of enzymes at a previous point in the pathway. In these circumstances, the biosynthetic intermediate before the break in the pathway often will be unable to serve as a precursor. Mutants of this tjqie normally do not survive unless provided with an outside source of an intermediate or product that occurs after the point at which the pathway is blocked. 

In some instances, an established intermediate may not be utilized by intact cells of mutants. Several obligatory intermediates of the shikimic acid pathway are not utilized when introduced exogenously. This phenomenon usually has been attributed to failure of the intermediate to reach the site of synthesis or utilization, a problem which sometimes can be averted with cell-free systems. The amount of a particular precursor introduced is also important. The presence of larger than normal amounts of an intermediate may also cause deviation from the usual sequence of events (Weiss and Edwards, 1981). 

The individual members of the shikimic acid pathway discussed above are examples of obligatory intermediates. The shikimic acid pathway is certainly the main route to the synthesis of the aromatic amino acids. Any doubts that might have existed have been removed by experiments with bacterial mutants. If, as a result of a mutation, the biosynthetic pathway is blocked beyond a certain substance and aromatic amino acids are then no longer formed, the substance affected must be an obligatory intermediate along the route to the synthesis of these amino acids. 

The usual method of study is to suggest a possible precursor and to feed it to the biosynthesizing system. The precursor has to be labelled in some way to trace it through the sequence of reactions, and that is usually by some isotopic element. It may be a radio-active isotope, such as H, " 0, or that can be followed by its radiation or it can be a stable heavy isotope, such as H, C, N, or 0, that can be traced by mass spectrometry or nuclear magnetic resonance (NMR) spectroscopy (Table 5.1). Another possible way is to use mutant strains of an organism that lack the enzymes to complete a particular synthesis, or to add a specific enzyme inhibitor, so that intermediates accumulate and can be identified. A mutant strain of yeast was important in discovering mevalonic acid and its place in terpene biosynthesis (Chapter 6) and a number of mutants of the bacterium Escherichia coli helped to understand the shikimic acid pathway (Chapter 8). 

The shikimate pathway was identified through the study of ultraviolet light-induced mutants of E. coli, Aerobacter aerogenes, and Neurospora. In 1950, using the penicillin enrichment technique (Chapter 26), Davis obtained a series of mutants of E. coli that would not grow without the addition of aromatic substances.4 5 A number of the mutants required five compounds tyrosine, phenylalanine, tryptophan, p-aminobenzoic acid, and a trace of p-hydroxybenzoic acid. It was a surprise to find that the requirements for all five compounds could be met by the addition of shikimic acid, an aliphatic compound that was then regarded as a rare plant acid. Thus, shikimate was implicated as an intermediate in the biosynthesis of the three aromatic amino acids and of other essential aromatic substances.6 7... 

The mutants that grew in the presence of shikimic acid evidently had the biosynthetic pathway blocked... 

The first two of these pathways were for many years generally postulated, on the basis of structural relations among various natural products and by analogy with known laboratory reactions they received experimental support later. The last pathway was discovered by B. D. Davis in his work with nutritionally deficient, microbial mutants. However, even earlier, when the structures of quinic acid (VII) and shikimic acid were established, their possible functions as intermediates in the biosynthesis of aromatic... 

Davis concluded that shikimic acid was a common precursor of phenylalanine, tyrosine, tryptophan, p-aminobenzoic acid, p-hydroxybenzoic acid, and an unknown sixth factor, and he next set out to determine other substances lying on the biosynthetic pathway. The various mutants were therefore tested for syntrophism, i.e., for the ability of one mutant to produce a substance necessary for the growth of another mutant. There was thus found a thermolabile substance, X, which was a true precursor of shikimic acid (184). X was isolated from culture filtrates and identified as 5-dehydroshikimic acid (744). Similar experiments revealed a substance, W, which was a true precursor of substance X (187, 193). This also was isolated and shown to be 5-dehydroquinic acid (906). The enzyme, named 5-dehydroquinase, converting dehydroquinic acid to dehydroshikimic acid has been partially purified (606). It is fairly stable, has a high specificity, appears to have no cofactors, and is of wide occurrence in bacteria, algae, yeasts, and plants but, as expected, could not be found in mammalian liver. 

In early examinations of the shUdmate pathway leading to aromatic compounds, a number of possible precursor compounds were evaluated. Surprisingly, out of about 50 compounds tested in mutant systems, only shikimic acid was found to be utilized. This compound could replace all three common aromatic amino acids and />-aminobenzoic acid (Weiss and Edwards, 1981). 

In the shikimic acid area, the conversion of D-glucose into the industrially important compound adipic acid using mutant enzymes derived from the shikimate pathway has been reported." ... 

Mutant strains of Escherichia coli and Aerobacter aerogenes were described which had a quintuple requirement of aromatic substrates (L-phenylalanine, L-tyrosine, L-tryptophan, 4-amino-benzoate and 4-hydroxybenzoate) for growth. Certain of these mutants were found to accumulate (—)-shikimic acid (4) in their culture filtrates and other mutants, blocked in earlier reactions in the pathway, were able to utilise (—)-shikimic acid (4) to replace the aromatic sutetrates. These observations established with great probability that (—)-shikimic add was a common precursor for each of these aromatic compounds. Experiments of this type permitted each of the intermediate in the common pathway, 3-dehydroquinic add (10), 3-dehydroshikimic add (11), (—)-shikimic add (4), shikimic add-3-phosphate (12), 5-enolpyruvylshikimic add-3-phosphate (13) and chorismic acid (14), to be isolated and characterised and for the pathway... 

The evidence that (- )-shikimic acid plays a central role in aromatic biosynthesis was obtained by Davis with a variety of nutritionally deficient mutants of Escherichia coli. In one group of mutants with a multiple requirement for L-tyrosine, L-phenylalanine, L-tryptophan and p-aminobenzoic acid and a partial requirement for p-hydroxybenzoic acid, (—)-shikimic acid substituted for all the aromatic compounds. The quintuple requirement for aromatic compounds which these mutants displayed arises from the fact that, besides furnishing a metabolic route to the three aromatic a-amino acids, the shikimate pathway also provides in micro-organisms a means of synthesis of other essential metabolites, and in particular, the various isoprenoid quinones involved in electron transport and the folic acid group of co-enzymes. The biosynthesis of both of these groups of compounds is discussed below. In addition the biosynthesis of a range of structurally diverse metabolites, which are derived from intermediates and occasionally end-products of the pathway, is outlined. These metabolites are restricted to certain types of organism and their function, if any, is in the majority of cases obscure. 

The identified intermediates in the aromatic biosynthetic pathway are ven in Fig. 12. Shikimic acid (3d>4a,5a-trihydroxy-A - -cyclohexene-1-carboxylic acid) was the first one to be isolated and identified, as already mentioned (iSOS). It was found to be the only compound that satisfied the total requirement for growth of the bacterial mutants exhibiting a quintuple aromatic requirement. The amount of shikimic acid producing... 

In a reaction that does not involve a monohydroxyphthalic acid, 4,5-dihy-droxyphthalic acid is formed. This is decarboxylated to protocatechuic acid the decarboxylation is unusual in that previously described reactions eliminating carboxyl groups from aromatic rings had all been oxidative. A biosynthesis involving a major metabolic pathway was indicated by the accumulation of protocatechuic acid by a Neurospora mutant blocked in the conversion of dihydroshikimic acid to shikimic acid (Gross et al, 1956). 

An mCyN unit is also present in the core of the polyketide antibiotic asukamycin from Streptomyces nodosas subsp. asukaensis [96]. This has been shown to arise not from a variant of the shikimate pathway, but from the condensation of a C4 unit from the TCA cycle, closely related to succinate, with a C3 unit, possibly dihydroxyacetone phosphate, from the triose pool. Related studies concerning 3-amino-4-hydroxybenzoic acid biosynthesis in Streptomyces murayamaensis mutants MC2 and MC3 support this hypothesis [97]. 

Use of Mutants in Biosynthetic Studies Formation of Chorismic Acid Derivatives of Chorismic Acid Biosynthesis of Tryptophan Indole 3-Acetic Acid Avenalumins from Oats DIMBOA and Related Compounds Biosynthesis of Phenylalanine and Tyrosine Compounds Derived from Shikimic Pathway Intermediates... 

The failure of 3-phosphoshikimic acid to be utilized by bacteria is in line with the general inability of intact cells to take up phosphate esters. Its utilization by cell-free extracts of suitable mutants shows it to be an essential intermediate in the common portion of the shikimate pathway. 

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